In vivo optical coherence tomography of light-driven melanosome translocation in retinal pigment epithelium

Abstract

Optical coherence tomography (OCT) may revolutionize fundamental investigation and clinical management of age-related macular degeneration and other eye diseases. However, quantitative OCT interpretation is hampered due to uncertain sub-cellular correlates of reflectivity in the retinal pigment epithelium (RPE) and photoreceptor. The purpose of this study was twofold: 1) to test OCT correlates in the RPE, and 2) to demonstrate the feasibility of longitudinal OCT monitoring of sub-cellular RPE dynamics. A high resolution OCT was constructed to achieve dynamic imaging of frog eyes, in which light-driven translocation of RPE melanosomes occurred within the RPE cell body and apical processes. Comparative histological examination of dark- and light-adapted eyes indicated that the RPE melanin granule, i.e., melanosome, was a primary OCT correlate. In vivo OCT imaging of RPE melanosomes opens the opportunity for quantitative assessment of RPE abnormalities associated with disease, and enables longitudinal investigation of RPE kinetics correlated with visual function.

Figure 1. Cartoon illustration of melanosome position in dark-adapted (left) and light-adapted (right) frog retinas.

RPE melanosomes are confined in the basal ends of the RPE cells in dark-adapted retinas; while they migrate into the apical projections in light-adapted retinas. ISe, inner segment ellipsoid.

Figure 2. Representative OCT images of dark-adapted and light-adapted frog eyes.

The cross-sectional images (A1) and (A2) were collected from two separate frogs, with over-night dark adaptation. NFL: nerve fiber layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ELM: external limiting membrane, IS: inner segment, OS: outer segment and RPE: retinal pigment epithelium. The cross-sectional images (B1) and (B2) were collected from two separate frogs, with 8-hour light adaptation. The green bars measured the retinal thickness with RPE complex included and the blue bars measured the RPE-ISe layer thickness; (C) Reconstructed en-face projection of blood vessels obtained nearby the NFL layer in Fig. 2A1; (D) Reconstructed en-face projection of photoreceptor mosaic obtained from the photoreceptor layer in Fig. 2A1. The contrast and brightness were adjusted for best visualization. (E) OCT reflectivity profiles of dark- (yellow) and light- (red) adapted frog eyes. The two yellow traces correspond to the two dark-adapted images in Fig. 2A1–2A2. The two red traces correspond to the two light-adapted images in Fig. 2B1–B2. Scale bars indicate 50 μm.

Figure 3. Box charts show RPE-ISe layer thickness (left) and retinal thickness (right) with data overlay.

Box = 25th and 75th percentile.

Figure 4. OCT imaging of retina during light-to-dark transition.

(A) Cross-sectional image of light-adapted frog eye; (B) Cross-sectional image at the identical location in the retina 1 hour later after maintained in the darkness. Panels (C–F) illustrate the same local areas of the cross-sectional images recorded at 0-min, 20-min, 40-min and 60-min after the light-adapted (A) retina was left in the darkness. Scale bars indicate 50 μm.

Figure 5. Light reflectivity change of inner and outer retina during light-to-dark transition.

(A) Red box indicates outer retina area with RPE complex included and green box indicates inner retina area; (B) the averaged reflectivity change of inner and outer retina during light-to-dark transition.

Figure 6. Histological images of dark- (A, C) and light-adapted (B, D) frog eyes.

For (A) and (B), images unstained 14 μm thick frozen sections were obtained by bright field microscopy (40×) with N.A. 0.95 objective; For (C) and (D), section thickness is 0.75 μm, and images were obtained by bright field microscopy (60×) with N.A. 1.4 objective. The inset to panel (D) shows individual melanosomes (arrows) within apical processes of RPE, flanked by photoreceptor outer segments, as obtained by transmission electron microscopy (6000× original magnification). Scale bars indicate 25 μm in (A–D) and 5 μm for (D) inset.

Figure 7. Schematic diagram of the experimental setup.

Schematic of the SD-OCT system at 846 nm designed to acquire in vivo images of the retina of the frog eye. SLD: superluminescent diode, PC: polarization controller, CL: collimation lens, L1–L5: lens. Focal lengths of lenses L1, L2, L3, L4, and L5 are 75, 50, 15, 100, and 30 mm, respectively. The photograph of the frog eye was taken by Qiu-Xiang Zhang.